SFS

source("../Rscripts/BaseScripts.R")
library(cowplot)
library(RColorBrewer)

1 Estiamte SFS with all individuals and all sites

(estimated by each chromosome and combine them later) (5.26.22~)

Step 1: Run PWS07_sfs_step1.sh (in “/Data/Slurumscripts/SFS_fromBam/PWS07_sfs_step1.sh”) for each population (takes a long time to create a saf file)

Step 2: Run PWS07_sfs_step2.sh to create unfolded sfs for each chromosome (Can’t run the whole genome due to memory constraints)

Step 2.2: Run PWS07_sfs_step2_folded.sh to create folded sfs for each chromosome

Step 3: Run R scripts to combine all sfs into 1

# at FARM, run the following scripts to combine sfs files
module load R
R
source("combineSFSfold.R") 
source("combineSFSunfold.R") 
combineSFSunfold("PWS07")   # this will create a "PWS07_unfolded.sfs" in /home/ktist/ph/data/angsd/SFS/fromBam/
combineSFSfold("PWS07")   # this will create a "PWS07_folded.sfs" in /home/ktist/ph/data/angsd/SFS/fromBam/folded/

#Exit R by typing 
quit()

Locally, you can run here:

combineSFSfold<-function(pop){
    ch1<-scan(paste0("Data/new_vcf/angsd/fromBam/folded/",pop,"_folded_chr1.sfs"))
    pws.sfs<-data.frame(chr1=ch1)
    for (i in 2:26){
        vec<-scan(paste0("Data/new_vcf/angsd/fromBam/folded/",pop,"_folded_chr",i,".sfs"))
        pws.sfs[,paste0("chr",i)]<-vec
    }
    pws.sfs$sum<-rowSums(pws.sfs)
    sink(paste0("Data/new_vcf/angsd/fromBam/folded/",pop,"_folded.sfs"))
    cat(pws.sfs$sum)
    sink(NULL)
}

combineSFSunfold<-function(pop){
    ch1<-scan(paste0("Data/new_vcf/angsd/fromBam/unfolded/",pop,"_unfolded_chr1.sfs"))
    pws.sfs<-data.frame(chr1=ch1)
    for (i in 2:26){
        vec<-scan(paste0("Data/new_vcf/angsd/fromBam/unfolded/",pop,"_unfolded_chr",i,".sfs"))
        pws.sfs[,paste0("chr",i)]<-vec
    }
    pws.sfs$sum<-rowSums(pws.sfs)
    sink(paste0("Data/new_vcf/angsd/fromBam/unfolded/",pop,"_unfolded.sfs"))
    cat(pws.sfs$sum)
    sink(NULL)
}

#Run with the 'pop' identifier
combineSFSunfold("PWS07")
combineSFSfold("PWS07")

Step 4: Run PWS07_sfs_theta.sh to calculate theta and Tajima’s D for unfolded.sfs (Fay & Wu’s H should be used with unfolded sfs)

Step 4.2: Run PWS07_sfs_step3_folded.sh to calculate theta and Tajima’s D for folded.sfs (Tajima’s D should be used with folded sfs)

1.1 SFS from bam files

1.1.1 1DSFS PWS

source("../Rscripts/BaseScripts.R")
pops<-c("PWS91","PWS96","PWS07","PWS17")

sfs1D<-data.frame()
for (i in 1:length(pops)){
    sfs <- scan(paste0("../Data/new_vcf/angsd/fromBam/combined/",pops[i],"_unfolded.sfs"))
    sfs1 <- data.frame(ac=sfs)
    sfs1$count<-0:(nrow(sfs1)-1)
    #remove the invariable sites
    sfs1<-sfs1[-c(1,nrow(sfs1)),]
    sfs1$pop<-pops[i]
    sfs1D<-rbind(sfs1D, sfs1)
}
    
sfs1D$pop<-factor(sfs1D$pop, levels=pops)
ggplot(data=sfs1D, aes(x=count, y=ac))+
    facet_wrap(~pop, ncol=4)+
        geom_bar(stat="identity", color="gray")+xlab("Frequency bin")+ ylab("Number of alleles")+
        theme_classic()+
        scale_y_continuous(labels=scales::comma)+
        theme(strip.background = element_rect(
            color="black", fill="gray80", size=0.5, linetype="solid"))
ggsave("../Output/SFS/1DSFS_fromBam_PWS.png", width = 11, height = 2.2, dpi=300)

1.1.2 Pi (π)

pops<-c("PWS91","PWS96","PWS07","PWS17")
theta<-data.frame()
for (i in 1:length(pops)){
    theta2<-read.delim(paste0('../Data/new_vcf/angsd/fromBam/unfolded/',pops[i],'_50kwin_10kstep.pestPG'))
    theta2$pi<-theta2$tP/theta2$nSites
    df<-theta2[,c("Chr","WinCenter","pi","fayh" )]
    df$pop<-pops[i]    
    theta<-rbind(theta, df)
}

#mean pi and Fay's H  (from unfolded SFS)
theta$pop<-factor(theta$pop, levels=pops)

ggplot(theta, aes(x=pop, y=pi))+
    geom_boxplot(position=position_dodge(width = 0.8), color=blu, outlier.alpha = 0.6,outlier.size = 0.7,width=0.6)+
    geom_point(stat = "summary", fun = "mean",position=position_dodge(width = 0.8))+
    ylab(expression(pi))+xlab("")+
    theme_bw()+
    theme(panel.grid.major.x = element_blank())+
    geom_vline(xintercept = c(1.5,2.5,3.5), color="gray", size=0.5)
ggsave("../Output/SFS/Pi_estimates_PWS_fromBam.png", width = 5, height = 3.5)

1.1.3 Fay’s H

ggplot(theta, aes(x=pop, y=fayh))+
    geom_boxplot(position=position_dodge(width = 0.8), color=grb, outlier.alpha = 0.6,outlier.size = 0.7,width=0.6)+
    geom_point(stat = "summary", fun = "mean",position=position_dodge(width = 0.8))+
    ylab("Fay's H")+xlab("")+
    theme_bw()+
    theme(panel.grid.major.x = element_blank())+
    geom_vline(xintercept = c(1.5,2.5,3.5), color="gray", size=0.5)
ggsave("../Output/SFS/FayH_estimates_PWS_fromBam.png", width = 5, height = 3.5)

1.1.4 Tajima’s D

#Tajima's D (folded SFS)
theta<-data.frame()
for (i in 1:length(pops)){
    theta2<-read.delim(paste0('../Data/new_vcf/angsd/fromBam/folded/',pops[i],'_50kwin_10kstep.pestPG'))
    df<-theta2[,c("Chr","WinCenter","Tajima" )]
    df$pop<-pops[i]    
    theta<-rbind(theta, df)
}

theta$pop<-factor(theta$pop, levels=pops)
ggplot(theta, aes(x=pop, y=Tajima))+
    geom_boxplot(position=position_dodge(width = 0.8), color=org, outlier.alpha = 0.6,outlier.size = 0.7,width=0.6)+
    geom_point(stat = "summary", fun = "mean",position=position_dodge(width = 0.8))+
    ylab("Tajima's D")+xlab("")+
    theme_bw()+
    theme(panel.grid.major.x = element_blank())+
    geom_vline(xintercept = c(1.5,2.5,3.5), color="gray", size=0.5)
ggsave("../Output/SFS/TajimaD_PWS_fromBam.png", width = 5, height = 3.5)

1.2 Estimate SFS from VCF files

(Using maf0.00 -no low allele freq cutoff)

Step 1: Run BC17_angsd_SFS.sh (in “/Data/Slurumscripts/SFS_fromVCFmaf00/”) for each population - this will calculate sfs, theta for both folded and unfolded SFS.

Step 2: Create 2D SFS by running each combination (ex. BC17CA172DSFS.sh in “/Data/Slurumscripts/SFS_fromVCFmaf00/”)

Step 3: Calculate Fst/Pbs for population combinations by running 3DFst scripts (ex. 3DFst_pws1.sh)

#Plot 2D SFS (Sfs_comparison.R)
source("../Rscripts/BaseScripts.R")

## 2D SFS
# The output from ANGSD is a flatten matrix: each value is the count of sites with the corresponding joint frequency ordered as
# [0,0] [0,1] [0,2] ..

# function to create a matrix from ANGSD output (a flatten matrix)
vec2mat<-function(vec, n1,n2, pop1, pop2){
    n1<-n1
    n2<-n2
    pop1<-pop1
    pop2<-pop2
    ANGSD.2D.SFS <- scan(paste(vec, sep=""), quiet=T)
    ANGSD.2D.SFS <- t(matrix(ANGSD.2D.SFS, nrow=n2*2+1, ncol=n1*2+1))
    # mask non-variant sites
    ANGSD.2D.SFS[1,1] <- 0
    ANGSD.2D.SFS[nrow(ANGSD.2D.SFS),ncol(ANGSD.2D.SFS)] <- 0
    df<-data.frame(ANGSD.2D.SFS)
    colnames(df)<-0:(ncol(df)-1)
    df$count<-0:(nrow(df)-1)
    return(df)
}

#Plot 2D SFS heatmap     
pops.info<-read.csv("../Data/Sample_metadata_892pops.csv")
pops.info$yr<-''
pops.info$yr[pops.info$year==96|pops.info$year==91]<-paste0(19,pops.info$year[pops.info$year==96|pops.info$year==91])
pops.info$yr[pops.info$year==07|pops.info$year==06|pops.info$year==17]<-paste0(20,pops.info$year[pops.info$year==07|pops.info$year==06|pops.info$year==17])
pops.info$yr<-apply(pops.info["yr"], 1, function(x) {if(x==206) x=2006
                                        if (x==207) x=2007
                                        else x=x})
pops.info$yr<-as.integer(pops.info$yr)
pops<-unique(pops.info$Population.Year)

pwss<-c("PWS91","PWS96","PWS07","PWS17")
tbs<-c("TB91","TB96","TB06","TB17")
sss<-c("SS96","SS06","SS17")
y17<-pops[grep("17",pops)]
comb1<-combn(pwss, 2)
comb1<-t(comb1)
comb2<-combn(tbs, 2)
comb2<-t(comb2)
comb3<-combn(sss, 2)
comb3<-t(comb3)
comb4<-combn(y17, 2)
comb4<-t(comb4)

#https://stackoverflow.com/questions/49689069/heatmap-with-continuous-rainbow-colours
cols <- rev(rainbow(7)[-7]) #rainbow colors for heatmap

#PWS
Plots<-list()
sfs.pws<-data.frame()
for (i in 1: nrow(comb1)){
    pop1<-comb1[i,1]
    pop2<-comb1[i,2]
    n1<-nrow(pops.info[pops.info$Population.Year==pop1,])
    n2<-nrow(pops.info[pops.info$Population.Year==pop2,])
    
    sfs1<-vec2mat(paste0("../Data/new_vcf/angsd/fromVCF/2D/folded_",pop1,"_",pop2,"_maf00.sfs"), n1=n1, n2=n2, pop1=pop1, pop2=pop2)
    
    if (pops.info$yr[pops.info$Population.Year==pop1][1]>pops.info$yr[pops.info$Population.Year==pop2][1]){
        sfs1<-data.frame(t(sfs1[,1:(ncol(sfs1)-1)]))
        colnames(sfs1)<-0:(ncol(sfs1)-1)
        sfs1$count<-0:(nrow(sfs1)-1)
        p2<-pop2
        pop2<-pop1
        pop1<-p2
    }
    #Plot first 30
    sfs2<-sfs1[1:30,1:30]
    sfs2$count<-0:(nrow(sfs2)-1)
    sfsm2<-melt(sfs2, id.vars="count")
    sfsm2$variable<-as.integer(as.character(sfsm2$variable))
    
    #zero as white (replace with NA)
    sfsm2$value[sfsm2$value==0]<-NA
    sfsm2$pop1<-pop1
    sfsm2$pop2<-pop2
    sfs.pws<-rbind(sfs.pws, sfsm2)
}

sfs.pws$pop1<-factor(sfs.pws$pop1, levels=c("PWS91","PWS96","PWS07","PWS17"))
sfs.pws$pop2<-factor(sfs.pws$pop2, levels=c("PWS91","PWS96","PWS07","PWS17"))

ggplot(sfs.pws, aes(x=count, y=variable))+
    facet_grid(pop2~pop1)+
    geom_raster(aes(fill=log10(value)))+xlab('')+ylab("")+
    scale_fill_gradientn(colors=cols, name="log(# of alleles)", na.value = "white")+
    theme_minimal()
ggsave("../Output/SFS/sfs_2D_PWS.png", width = 10, height = 8, dpi=300)
   

#TB
sfs.tb<-data.frame()
for (i in 1: nrow(comb2)){
    pop1<-comb2[i,1]
    pop2<-comb2[i,2]
    n1<-nrow(pops.info[pops.info$Population.Year==pop1,])
    n2<-nrow(pops.info[pops.info$Population.Year==pop2,])
    
    sfs1<-vec2mat(paste0("../Data/new_vcf/angsd/fromVCF/2D/folded_",pop1,"_",pop2,"_maf00.sfs"), n1=n1, n2=n2, pop1=pop1, pop2=pop2)
    
    if (pops.info$yr[pops.info$Population.Year==pop1][1]>pops.info$yr[pops.info$Population.Year==pop2][1]){
        sfs1<-data.frame(t(sfs1[,1:(ncol(sfs1)-1)]))
        colnames(sfs1)<-0:(ncol(sfs1)-1)
        sfs1$count<-0:(nrow(sfs1)-1)
        p2<-pop2
        pop2<-pop1
        pop1<-p2
    }
    #Plot first 30
    sfs2<-sfs1[1:30,1:30]
    sfs2$count<-0:(nrow(sfs2)-1)
    sfsm2<-melt(sfs2, id.vars="count")
    sfsm2$variable<-as.integer(as.character(sfsm2$variable))
    
    #zero as white (replace with NA)
    sfsm2$value[sfsm2$value==0]<-NA
    sfsm2$pop1<-pop1
    sfsm2$pop2<-pop2
    sfs.tb<-rbind(sfs.tb, sfsm2)
}

sfs.tb$pop1<-factor(sfs.tb$pop1, levels=c("TB91","TB96","TB06","TB17"))
sfs.tb$pop2<-factor(sfs.tb$pop2, levels=c("TB91","TB96","TB06","TB17"))

ggplot(sfs.tb, aes(x=count, y=variable))+
    facet_grid(pop2~pop1)+
    geom_raster(aes(fill=log10(value)))+xlab('')+ylab("")+
    scale_fill_gradientn(colors=cols, name="log(# of alleles)", na.value = "white")+
    theme_minimal()
ggsave("../Output/SFS/sfs_2D_TB.png", width = 10, height = 8, dpi=300)

#SS
sfs.ss<-data.frame()
for (i in 1: nrow(comb3)){
    pop1<-comb3[i,1]
    pop2<-comb3[i,2]
    n1<-nrow(pops.info[pops.info$Population.Year==pop1,])
    n2<-nrow(pops.info[pops.info$Population.Year==pop2,])
    sfs1<-vec2mat(paste0("../Data/new_vcf/angsd/fromVCF/2D/folded_",pop1,"_",pop2,"_maf00.sfs"), n1=n1, n2=n2, pop1=pop1, pop2=pop2)
    if (pops.info$yr[pops.info$Population.Year==pop1][1]>pops.info$yr[pops.info$Population.Year==pop2][1]){
        sfs1<-data.frame(t(sfs1[,1:(ncol(sfs1)-1)]))
        colnames(sfs1)<-0:(ncol(sfs1)-1)
        sfs1$count<-0:(nrow(sfs1)-1)
        p2<-pop2
        pop2<-pop1
        pop1<-p2
    }
    #Plot first 30
    sfs2<-sfs1[1:30,1:30]
    sfs2$count<-0:(nrow(sfs2)-1)
    sfsm2<-melt(sfs2, id.vars="count")
    sfsm2$variable<-as.integer(as.character(sfsm2$variable))
    
    #zero as white (replace with NA)
    sfsm2$value[sfsm2$value==0]<-NA
    sfsm2$pop1<-pop1
    sfsm2$pop2<-pop2
    sfs.ss<-rbind(sfs.ss, sfsm2)
}
sfs.ss$pop1<-factor(sfs.ss$pop1, levels=c("SS96","SS06","SS17"))
sfs.ss$pop2<-factor(sfs.ss$pop2, levels=c("SS96","SS06","SS17"))

ggplot(sfs.ss, aes(x=count, y=variable))+
    facet_grid(pop2~pop1)+
    geom_raster(aes(fill=log(value)))+xlab('')+ylab("")+
    scale_fill_gradientn(colors=cols, name="log(# of alleles)", na.value = "white")+
    theme_minimal()
ggsave("../Output/SFS/sfs_2D_SS.png", width = 10, height = 8, dpi=300)

#2017 pops
sfs17<-data.frame()
for (i in 1: nrow(comb4)){
    pop1<-comb4[i,1]
    pop2<-comb4[i,2]
    n1<-nrow(pops.info[pops.info$Population.Year==pop1,])
    n2<-nrow(pops.info[pops.info$Population.Year==pop2,])
    sfs1<-vec2mat(paste0("../Data/new_vcf/angsd/fromVCF/2D/folded_",pop1,"_",pop2,"_maf00.sfs"), n1=n1, n2=n2, pop1=pop1, pop2=pop2)
    
    #Plot first 30
    sfs2<-sfs1[1:30,1:30]
    sfs2$count<-0:(nrow(sfs2)-1)
    sfsm2<-melt(sfs2, id.vars="count")
    sfsm2$variable<-as.integer(as.character(sfsm2$variable))
    
    #zero as white (replace with NA)
    sfsm2$value[sfsm2$value==0]<-NA
    sfsm2$pop1<-pop1
    sfsm2$pop2<-pop2
    sfs17<-rbind(sfs17, sfsm2)
}

sfs17$pop1<-factor(sfs17$pop1, levels=paste(y17))
sfs17$pop2<-factor(sfs17$pop2, levels=paste(y17))

ggplot(sfs17, aes(x=count, y=variable))+
    facet_grid(pop2~pop1)+
    geom_raster(aes(fill=log(value)))+xlab('')+ylab("")+
    scale_fill_gradientn(colors=cols, name="log(# of alleles)", na.value = "white")+
    theme_minimal()
ggsave("../Output/SFS/sfs_2D_2017.png", width = 20, height = 16, dpi=300)

1.2.1 1D SFS all populations (Downsampled bam files)

1.2.2 2D SFS PWS

1.2.3 2D SFS TB

1.2.4 2D SFS SS

1.2.5 2D SFS (2017)

2 Fst between years per population

2.1 PWS

2.1.1 Fst along the genome

### Fst estimates from 2Dsfs
pops<-c("PWS91","PWS96","PWS07","PWS17")
comb<-t(combn(pops,2))

fst<-data.frame()
for (i in 1: nrow(comb)){
    pop1<-comb[i,1]
    pop2<-comb[i,2]
    df<-read.delim(paste0("../Data/new_vcf/angsd/fromVCF/2D/fst_",pop1,"_",pop2,"_50kWindow_maf00"))
    conames<-colnames(df)[2:4]
    colnames(df)[4]<-"Fst"
    colnames(df)[1:3]<-conames
    df$pop<-paste0(pop1,".vs.",pop2)
    df$ch=as.integer(gsub("chr","", df$chr))
    df<-df[order(df$ch),]
    df$loc<-1:nrow(df)
    fst<-rbind(fst, df)
}

evens<-paste0("chr",seq(2,26, by=2))
#Plot Fst valuese across Genome
fst$color<-"col1"
fst$color[fst$chr %in% evens]<-"col2"
fst$pop<-factor(fst$pop, levels=unique(fst$pop))

#add chromosome number
df<-fst[fst$pop=="PWS91.vs.PWS96",]
rows<-data.frame(chr=1:26)
for (i in 1:26){
    if (i ==1){
        rows$n[i]<-nrow(df[df$ch==i,])
        rows$middle[i]<--nrow(df[df$ch==i,])/2
    }
    if (i >1){
        rows$n[i]<-nrow(df[df$ch==i,])
        rows$middle[i]<-sum(rows$n[1:(i-1)])+rows$n[i]/2
    }
}
#
ggplot(fst, aes(x=loc, y=Fst, color=color))+
    facet_wrap(~pop, ncol = 1, strip.position="right")+
    geom_point(size=0.2)+
    scale_color_manual(values=c("gray50","steelblue"))+
    theme_bw()+
    ylab("Fst")+xlab('Genome position')+
    theme(legend.position = "none", panel.grid.major.x = element_blank(), panel.grid.minor.x = element_blank())+
    scale_x_continuous(breaks=rows$middle, labels=1:26)
ggsave("../Output/SFS/PWS_Fst_pairwise_comparison.png", width = 20, height = 10, dpi=150)

### Pairwise Fst along each chromosome

## Plot Fst values along each chromosome
fst$chr<-factor(fst$chr, levels=paste0("chr",1:26))
fstpw<-fst
plots<-list()
compare<-paste0(unique(fstpw$pop))
max(fstpw$Fst)
for (i in 1:6){ 
    fs<-gsub("vs.","",compare[i])
    pops <- unlist(strsplit(fs, "\\."))
    maxy<-max(fstpw$Fst[fstpw$pop==compare[i]])
    # Fst with actual line to highlight the differences
    plots[[i]] <- ggplot(fstpw[fstpw$pop==compare[i],], aes(x =midPos, y =Fst )) + 
        geom_point(size = 1, color = gry,alpha = 0.4, shape = 1)+
        theme_minimal()+ylim(0,maxy+0.02)+
        theme(axis.text.x=element_blank())+
        ylab("Fst\n")+ xlab("")+ 
        ggtitle(paste0(pops[1]," vs.", pops[2]))+
        geom_line(color=blu, size=0.2)+
        facet_wrap(~chr, ncol = 9)
}
#save the plots together
{png(paste0("../Output/SFS/PWS_Fst_maf00_chr.png"), height = 8, width = 18, res=150, units = "in")
grid.arrange(plots[[3]], plots[[2]], plots[[4]], plots[[1]],plots[[5]],plots[[6]], ncol=3)
dev.off()}

Pairwise Fst for each genome

2.1.2 Create pairwise Fst matrix

## ..continued from above 
pops<-c("PWS91","PWS96","PWS07","PWS17")
comb<-t(combn(pops,2))

## Plot average fst in a heatmap
comp<-unique(fst$pop)
pairfst<-data.frame(matrix(ncol=4, nrow=4), row.names=pops)
colnames(pairfst)<-pops
for (i in 1:6){
    pop1<-comb[i,1]
    pop2<-comb[i,2]
    df<-fst[fst$pop==comp[i],]
    pairfst[pop1,pop2]<-mean(df$Fst, na.rm=T)
    #pairfst[pop2,pop1]<-mean(df$Fst, na.rm=T)
}
write.csv(pairfst,"../Output/SFS/PWS_pairwiseFst_matrix.csv")

pairfst<-read.csv("../Output/SFS/PWS_pairwiseFst_matrix.csv", row.names = 1)
df<-pairfst
diag(df)<-0
df$pop<-rownames(df)
dfm<-melt(df,na.rm=T, id.vars='pop')

#NA to diagonal
dfm$value[dfm$value==0]<-NA
dfm$pop<-factor(dfm$pop, levels=pops)
dfm$value<-round(dfm$value, 4)
ggplot(data = dfm, aes(pop, variable, fill = value))+
    geom_tile(color = "white")+
    scale_fill_gradientn(colors=c("white", "#0C54FF"), limits=c(0, (max(dfm$value, na.rm=T)+0.005)),na.value="gray80", 
                         name="Fst")+
    theme_minimal()+ xlab("")+ylab("")+
    theme(axis.text.x = element_text(angle = 0, vjust = 0, 
                                     size = 12, hjust = 0.5))+
    theme(axis.text.y = element_text(size = 12))+
    coord_fixed()+
    geom_text(aes(pop, variable, label = value), color = "black", size = 5)
ggsave(paste0("../Output/SFS/pairwiseFst_PWS.png"), width = 5, height = 5, dpi=150)

PWS Pairwise Fst

2.1.3 Fst change over time

# Plot Fst in a bar plot ordered and colored in the same way as Fst/Pi shuffle results (Shuffling_pi.fst.tehat.Rmd)
fsts<-dfm[!is.na(dfm$value),]
fsts$comp<-paste0(fsts$pop,"_",fsts$variable)

#set the colors
div1<-diverging_hcl(6, palette="Blue-Red")
div2<-rev(div1)
names(div2)<-c("PWS96_PWS07","PWS07_PWS17","PWS91_PWS96", "PWS91_PWS07", "PWS91_PWS17","PWS96_PWS17")
fsts<-fsts[order(fsts$value, decreasing = T),]
fsts$comp<-factor(fsts$comp, levels=paste0(unique(fsts$comp)))

ggplot(fsts, aes(x=comp, y=value, fill=comp))+
    geom_bar(stat="identity")+
    scale_fill_manual(name = "comp",values = div2)+
    xlab('')+ylab('Fst')+
    theme_classic()+
    theme(axis.text.x = element_text(angle=45, hjust=1), legend.title = element_blank())
ggsave("../Output/Fst/PairwiseFst_ordered.png", width = 4, height = 2.8, dpi=300 )

#Fst over time

fsts2<-fsts[fsts$comp %in% c("PWS91_PWS96","PWS96_PWS07","PWS07_PWS17"),]
fsts2$time<-1
fsts2$time[fsts2$comp=="PWS96_PWS07"]<-2
fsts2$time[fsts2$comp=="PWS07_PWS17"]<-3

ggplot(fsts2, aes(x=time, y=value))+
    geom_point(size=3, color="steelblue")+
    geom_path(color="steelblue")+
    theme_classic()+ylab("Fst")+xlab("")+
    scale_x_continuous(breaks=c(1,2,3), labels=c("91-96", "96-07","07-17"))
ggsave("../Output/Fst/Fst_overTime.png", width = 4, height = 2.8, dpi=300 )

fsts$series<-"1991-2007, 1991-2017"
fsts$series[fsts$comp %in% c("PWS91_PWS96","PWS96_PWS07","PWS07_PWS17")]<-"1991-1996, 1996-2007, 2007-2017"
fsts$series[fsts$comp =="PWS96_PWS17"]<-"1996-2017"
fsts$time<-1
fsts$time[fsts$variable=="PWS17"]<-3
fsts$time[fsts$variable=="PWS07"]<-2
source("../Rscripts/BaseScripts.R")

ggplot(fsts, aes(x=time, y=value, color=series))+
    geom_point(size=3)+
    geom_path()+
    scale_color_manual(values=cols)+
    theme_classic()+ylab("Fst")+xlab("")+
    scale_x_continuous(breaks=c(1,2,3), labels=c("1991-1996", "~2007","~2017"))+
    theme(legend.title = element_blank())
ggsave("../Output/Fst/Fst_overTime_allComparison.png", width = 6, height = 2.8, dpi=300 )


fstP1<-fsts
fstP2<-fsts2

2.1.4 Plot mean Fst for each chromosome

# Plot mean Fst of each chromosme
Fst<-data.frame()
compare<-paste0(unique(fstpw$pop))
for (j in 1:26){
    fst.ch<-fst[fst$ch==j,]
    pairch<-data.frame(matrix(ncol=4, nrow=4), row.names=pops)
    colnames(pairch)<-pops
    for (i in 1:6){
        pop1<-comb[i,1]
        pop2<-comb[i,2]
        df<-fst.ch[fst.ch$pop==compare[i],]
        pairch[pop1,pop2]<-mean(df$Fst, na.rm=T)
    }
    diag(pairch)<-0
    pairch$pop<-rownames(pairch)
    dfm<-melt(pairch,na.rm=T, id.vars='pop')
    
    #NA to diagonal
    dfm$value[dfm$value==0]<-NA
    dfm$pop<-factor(dfm$pop, levels=pops)
    dfm$value<-round(dfm$value, 4)
    dfm$chr<-j
    Fst<-rbind(Fst, dfm)
    
}
Fst$id<-paste0(Fst$pop," vs.",Fst$variable)
Fst<-Fst[!is.na(Fst$value),]
ggplot(Fst, aes(x=chr, y=value,color=id))+
    geom_point()+
    geom_path(stat="identity")+
    theme_minimal()+ylab("Fst")+
    scale_x_continuous(breaks=1:26, labels = 1:26)+
    theme(legend.title = element_blank(), panel.grid.minor.x = element_blank())
ggsave("../Output/SFS/PWS_Fst_byChromosome_dotplot.png", width = 8, height=4.5, dpi=150)

PWS average Fst per chromosome

2.2 Togiak Bay

2.2.1 Pairwise Fst along the genome

2.2.2 Pairwise Fst along each chromosome

#To save the plots, run in R
png(paste0("Output/SFS/TB_Fst_maf00_chr.png"), height = 8, width = 18, res=150, units = "in")  
grid.arrange(plots[[3]], plots[[2]], plots[[4]], plots[[1]],plots[[5]],plots[[6]], ncol=3)  
dev.off()  

2.2.3 Pairwise Fst matrix

## Continued from the above
pops<-c("TB91","TB96","TB06","TB17")
comb<-t(combn(pops,2))

## Plot average fst in a heatmap
compare<-unique(fst$pop)
pairfst<-data.frame(matrix(ncol=4, nrow=4), row.names=pops)
colnames(pairfst)<-pops
for (i in 1:6){
    pop1<-comb[i,1]
    pop2<-comb[i,2]
    df<-fst[fst$pop==compare[i],]
    pairfst[pop1,pop2]<-mean(df$Fst, na.rm=T)
}
write.csv(pairfst,"../Output/SFS/TB_pairwiseFst_matrix.csv")

pairfst<-read.csv("../Output/SFS/TB_pairwiseFst_matrix.csv", row.names = 1)

df<-pairfst
diag(df)<-0
df$pop<-rownames(df)
dfm<-melt(df,na.rm=T, id.vars='pop')
#NA to diagonal
dfm$value[dfm$value==0]<-NA
dfm$pop<-factor(dfm$pop, levels=pops)
dfm$value<-round(dfm$value, 4)
ggplot(data = dfm, aes(pop, variable, fill = value))+
    geom_tile(color = "white")+
    scale_fill_gradientn(colors=c("white", "#0C54FF"), limits=c(0, (max(dfm$value, na.rm=T)+0.005)),na.value="gray80", 
                         name="Fst")+
    theme_minimal()+ xlab("")+ylab("")+
    theme(axis.text.x = element_text(angle = 0, vjust = 0, 
                                     size = 12, hjust = 0.5))+
    theme(axis.text.y = element_text(size = 12))+
    coord_fixed()+
    geom_text(aes(pop, variable, label = value), color = "black", size = 5)
ggsave(paste0("../Output/SFS/pairwiseFst_TB.png"), width = 5, height = 5, dpi=150)

2.2.4 Fst change over time

# Plot Fst in a bar plot ordered and colored in the same way as Fst/Pi shuffle results (Shuffling_pi.fst.tehat.Rmd)
fsts<-dfm[!is.na(dfm$value),]
fsts$comp<-paste0(fsts$pop,"_",fsts$variable)

#set the colors
#div1<-diverging_hcl(6, palette="Blue-Red")
#div2<-rev(div1)
#names(div2)<-c("PWS96_PWS07","PWS07_PWS17","PWS91_PWS96", "PWS91_PWS07", "PWS91_PWS17","PWS96_PWS17")
fsts<-fsts[order(fsts$value, decreasing = T),]
fsts$comp<-factor(fsts$comp, levels=paste0(unique(fsts$comp)))

ggplot(fsts, aes(x=comp, y=value, fill=comp))+
    geom_bar(stat="identity")+
    scale_fill_manual(values = div1)+
    xlab('')+ylab('Fst')+
    theme_classic()+
    theme(axis.text.x = element_text(angle=45, hjust=1), legend.title = element_blank())
ggsave("../Output/Fst/TB_PairwiseFst_ordered.png", width = 4, height = 2.8, dpi=300 )

#Fst over time
fsts2<-fsts[fsts$comp %in% c("TB91_TB96","TB96_TB06","TB06_TB17"),]
fsts2$time<-1
fsts2$time[fsts2$comp=="TB96_TB06"]<-2
fsts2$time[fsts2$comp=="TB06_TB17"]<-3
fsts2<-fsts2[order(fsts2$time),]
ggplot(fsts2, aes(x=time, y=value))+
    geom_point(size=3, color="steelblue")+
    geom_path( aes(x=time, y=value),color="steelblue")+
    theme_classic()+ylab("Fst")+xlab("")+
    scale_x_continuous(breaks=c(1,2,3), labels=c("1991-1996", "1996-2006","2006-2017"))
ggsave("../Output/Fst/Fst_overTime_TB.png", width = 4, height = 2.8, dpi=300 )

fsts$series<-"1991-2007, 1991-2017"
fsts$series[fsts$comp %in% c("TB91_TB96","TB96_TB06","TB06_TB17")]<-"1991-1996, 1996-2007, 2007-2017"
fsts$series[fsts$comp =="TB96_TB17"]<-"1996-2017"
fsts$time<-1
fsts$time[fsts$variable=="TB17"]<-3
fsts$time[fsts$variable=="TB06"]<-2
#source("../Rscripts/BaseScripts.R")
fsts<-fsts[order(fsts$time),]
ggplot(fsts, aes(x=time, y=value, color=series))+
    geom_point(size=3)+
    geom_path()+
    scale_color_manual(values=cols)+
    theme_classic()+ylab("Fst")+xlab("")+
    scale_x_continuous(breaks=c(1,2,3), labels=c("1991-1996", "~2007","~2017"))+
    theme(legend.title = element_blank())
ggsave("../Output/Fst/Fst_overTime_TB_allComparison.png", width = 6, height = 2.8, dpi=300 )

# plot both PWS and TB together

fstP2$pop<-"PWS"
fsts2$pop<-"TB"
fstPT<-rbind(fstP2, fsts2)

ggplot(fstPT, aes(x=time, y=value, color=pop))+
    geom_point(size=3)+
    geom_path( aes(x=time, y=value))+
    scale_color_manual(values=cols[c(2,1)])+
    theme_classic()+ylab("Fst")+xlab("")+
    scale_x_continuous(breaks=c(1,2,3), labels=c("1991-1996", "1996-2006","2006-2017"))+
    theme(legend.title = element_blank())
ggsave("../Output/Fst/Fst_overTime_PWS_TB.png", width = 4, height = 2.8, dpi=300 )

fstT1<-fsts
fstT2<-fsts2

2.2.5 Mean pairsie Fst per chromosome

# Plot mean Fst of each chromosme
Fst<-data.frame()
for (j in 1:26){
    fst.ch<-fst[fst$ch==j,]
    pairch<-data.frame(matrix(ncol=4, nrow=4), row.names=pops)
    colnames(pairch)<-pops
    for (i in 1:6){
        pop1<-comb[i,1]
        pop2<-comb[i,2]
        df<-fst.ch[fst.ch$pop==compare[i],]
        pairch[pop1,pop2]<-mean(df$Fst, na.rm=T)
    }
    diag(pairch)<-0
    pairch$pop<-rownames(pairch)
    dfm<-melt(pairch,na.rm=T, id.vars='pop')
    
    #NA to diagonal
    dfm$value[dfm$value==0]<-NA
    dfm$pop<-factor(dfm$pop, levels=pops)
    dfm$value<-round(dfm$value, 4)
    dfm$chr<-j
    Fst<-rbind(Fst, dfm)
    
}
Fst$id<-paste0(Fst$pop," vs.",Fst$variable)
Fst<-Fst[!is.na(Fst$value),]
ggplot(Fst, aes(x=chr, y=value,color=id))+
    geom_point()+
    geom_path(stat="identity")+
    theme_minimal()+ylab("Fst")+
    scale_x_continuous(breaks=1:26, labels = 1:26)+
    theme(legend.title = element_blank(), panel.grid.minor.x = element_blank())
ggsave("../Output/SFS/TB_Fst_byChromosome_dotplot.png", width = 13, height=6.5, dpi=150)

TB mean Fst per chromosome

2.3 Sitka Sound

2.3.1 Pairwise Fst along the genome

2.3.2 Pairwise Fst along each chromosome

## Plot Fst values along each chromosome
fst$chr<-factor(fst$chr, levels=paste0("chr",1:26))
fstss<-fst
plots<-list()
compare<-paste0(unique(fstss$pop))
for (i in 1:3){ 
    fs<-gsub("vs.","",compare[i])
    pops <- unlist(strsplit(fs, "\\."))
    maxy<-max(fstss$Fst[fstss$pop==compare[i]])
    # Fst with actual line to highlight the differences
    plots[[i]] <- ggplot(fstss[fstss$pop==compare[i],], aes(x =midPos, y =Fst )) + 
        geom_point(size = 1, color = gry,alpha = 0.4, shape = 1)+
        theme_minimal()+ylim(0,maxy+0.02)+
        theme(axis.text.x=element_blank())+
        ylab("Fst\n")+ xlab("")+ 
        ggtitle(paste0(pops[1]," vs.", pops[2]))+
        geom_line(color=blu, size=0.2)+
        facet_wrap(~chr, ncol = 9)
}

{png(paste0("Output/SFS/SS_Fst_maf00_chr.png"), height = 4, width = 18, res=150, units = "in")  
grid.arrange(plots[[1]], plots[[2]], plots[[3]], ncol=3)  
dev.off()  }

2.3.3 Pairwise Fst matrix

## Continued from the above
pops<-c("SS96","SS06","SS17")
comb<-t(combn(pops,2))

## Plot average fst in a heatmap
compare<-unique(fst$pop)
pairfst<-data.frame(matrix(ncol=3, nrow=3), row.names=pops)
colnames(pairfst)<-pops
for (i in 1:3){
    pop1<-comb[i,1]
    pop2<-comb[i,2]
    df<-fst[fst$pop==compare[i],]
    pairfst[pop1,pop2]<-mean(df$Fst, na.rm=T)
}
write.csv(pairfst,"../Output/SFS/SS_pairwiseFst_matrix.csv")

pairfst<-read.csv("../Output/SFS/SS_pairwiseFst_matrix.csv", row.names = 1)
df<-pairfst
diag(df)<-0
df$pop<-rownames(df)
dfm<-melt(df,na.rm=T, id.vars='pop')
#NA to diagonal
dfm$value[dfm$value==0]<-NA
dfm$pop<-factor(dfm$pop, levels=pops)
dfm$value<-round(dfm$value, 4)
ggplot(data = dfm, aes(pop, variable, fill = value))+
    geom_tile(color = "white")+
    scale_fill_gradientn(colors=c("white", "#0C54FF"), limits=c(0, (max(dfm$value, na.rm=T)+0.005)),na.value="gray80", 
                         name="Fst")+
    theme_minimal()+ xlab("")+ylab("")+
    theme(axis.text.x = element_text(angle = 0, vjust = 0, 
                                     size = 12, hjust = 0.5))+
    theme(axis.text.y = element_text(size = 12))+
    coord_fixed()+
    geom_text(aes(pop, variable, label = value), color = "black", size = 5)
ggsave(paste0("../Output/SFS/pairwiseFst_SS.png"), width = 5, height = 5, dpi=150)

2.3.4 Fst change over time

# Plot Fst in a bar plot ordered and colored in the same way as Fst/Pi shuffle results (Shuffling_pi.fst.tehat.Rmd)
fsts<-dfm[!is.na(dfm$value),]
fsts$comp<-paste0(fsts$pop,"_",fsts$variable)

#set the colors
#div1<-diverging_hcl(6, palette="Blue-Red")
#div2<-rev(div1)
#names(div2)<-c("PWS96_PWS07","PWS07_PWS17","PWS91_PWS96", "PWS91_PWS07", "PWS91_PWS17","PWS96_PWS17")
fsts<-fsts[order(fsts$value, decreasing = T),]
fsts$comp<-factor(fsts$comp, levels=paste0(unique(fsts$comp)))

ggplot(fsts, aes(x=comp, y=value, fill=comp))+
    geom_bar(stat="identity")+
    scale_fill_manual(values = div1)+
    xlab('')+ylab('Fst')+
    theme_classic()+
    theme(axis.text.x = element_text(angle=45, hjust=1), legend.title = element_blank())
ggsave("../Output/Fst/TB_PairwiseFst_ordered.png", width = 3.4, height = 2.8, dpi=300 )

#Fst over time
fsts2<-fsts[fsts$comp %in% c("SS96_SS06","SS06_SS17"),]
fsts2$time<-1
fsts2$time[fsts2$comp=="SS96_SS06"]<-2
fsts2$time[fsts2$comp=="SS06_SS17"]<-3
fsts2<-fsts2[order(fsts2$time),]
ggplot(fsts2, aes(x=time, y=value))+
    geom_point(size=3, color="steelblue")+
    geom_path( aes(x=time, y=value),color="steelblue")+
    theme_classic()+ylab("Fst")+xlab("")+
    scale_x_continuous(breaks=c(1,2,3), labels=c("1991-1996", "1996-2006","2006-2017"))
ggsave("../Output/Fst/Fst_overTime_SS.png", width = 3.5, height = 2.8, dpi=300 )

fsts$series<-"1991-2007, 1991-2017"
fsts$series[fsts$comp =="SS96_SS17"]<-"1996-2017"
fsts$time<-1
fsts$time[fsts$variable=="SS17"]<-3
fsts$time[fsts$variable=="SS06"]<-2
#source("../Rscripts/BaseScripts.R")
fsts<-fsts[order(fsts$time),]
ggplot(fsts, aes(x=time, y=value, color=series))+
    geom_point(size=3)+
    geom_path()+
    scale_color_manual(values=cols)+
    theme_classic()+ylab("Fst")+xlab("")+
    scale_x_continuous(breaks=c(1,2,3), labels=c("1991-1996", "~2007","~2017"))+
    theme(legend.title = element_blank())
ggsave("../Output/Fst/Fst_overTime_SS_allComparison.png", width = 6, height = 2.8, dpi=300 )


# plot all 3 pops together

fstPTS<-rbind(fstPT,fsts2)
write.csv(fstPTS, "../Output/Fst/Mean_Fst_overTime_3pops.csv", row.names = F)


fstPST<-read.csv("../Output/Fst/Mean_Fst_overTime_3pops.csv")

ggplot(fstPST, aes(x=time, y=value, color=pop))+
    geom_point(size=3)+
    geom_path( aes(x=time, y=value))+
    scale_color_manual(values=cols[c(2,1,3)])+
    theme_classic()+ylab("Fst")+xlab("")+
    scale_x_continuous(breaks=c(1,2,3), labels=c("1991-1996", "1996-2006","2006-2017"))+
    theme(legend.title = element_blank())
ggsave("../Output/Fst/Fst_overTime_3Pops.png", width = 5, height = 2.8, dpi=300 )

fstP1$pop<-"PWS"
fstT1$pop<-"TB"
fsts$pop<-"SS"
fsts3<-rbind(fstP1, fstT1, fsts)
write.csv(fsts3, "../Output/Fst/Mean_Fst_overTime_3pops_allcomp.csv")

2.3.5 Mean pairsie Fst per chromosome

# Plot mean Fst of each chromosomes
Fst<-data.frame()
for (j in 1:26){
    fst.ch<-fst[fst$ch==j,]
    pairch<-data.frame(matrix(ncol=3, nrow=3), row.names=pops)
    colnames(pairch)<-pops
    for (i in 1:3){
        pop1<-comb[i,1]
        pop2<-comb[i,2]
        df<-fst.ch[fst.ch$pop==compare[i],]
        pairch[pop1,pop2]<-mean(df$Fst, na.rm=T)
    }
    diag(pairch)<-0
    pairch$pop<-rownames(pairch)
    dfm<-melt(pairch,na.rm=T, id.vars='pop')
    
    #NA to diagonal
    dfm$value[dfm$value==0]<-NA
    dfm$pop<-factor(dfm$pop, levels=pops)
    dfm$value<-round(dfm$value, 4)
    dfm$chr<-j
    Fst<-rbind(Fst, dfm)
    
}
Fst$id<-paste0(Fst$pop," vs.",Fst$variable)
Fst<-Fst[!is.na(Fst$value),]
ggplot(Fst, aes(x=chr, y=value,color=id))+
    geom_point()+
    geom_path(stat="identity")+
    theme_minimal()+ylab("Fst")+
    scale_x_continuous(breaks=1:26, labels = 1:26)+
    theme(legend.title = element_blank(), panel.grid.minor.x = element_blank())
ggsave("../Output/SFS/SS_Fst_byChromosome_dotplot.png", width = 8, height=4.5, dpi=150)

3 Fst between populations (2017)

3.1 Pairiwse Fst along the genome

popsn<-read.csv("../Data/Sample_metadata_892pops.csv")
pops<-unique(popsn$Population.Year)
y17<-pops[grep("17",pops)]

comb<-combn(y17, 2)
comb<-t(comb)

#Year2017
fst17<-data.frame()
for (i in 1: nrow(comb)){
    pop1<-comb[i,1]
    pop2<-comb[i,2]
    df<-read.delim(paste0("../Data/new_vcf/angsd/fromVCF/2D/fst_folded_",pop1,"_",pop2,"_50kWindow_maf00"))
    conames<-colnames(df)[2:4]
    colnames(df)[4]<-"Fst"
    colnames(df)[1:3]<-conames
    df$pop<-paste0(pop1,".vs.",pop2)
    df$ch=as.integer(gsub("chr","", df$chr))
    df<-df[order(df$ch),]
    df$loc<-1:nrow(df)
    fst17<-rbind(fst17, df)
}

write.csv(fst17,"../Output/SFS/Fst_window_year2017_allpops.csv")

fst17$ch<-as.integer(gsub("chr","",fst17$chr))
fst17<-fst17[order(fst17$ch, fst17$midPos),]
fst17$chr<-factor(fst17$chr, levels=paste0("chr",1:26))

pairs<-unique(fst17$pop)
plots<-list()
for (i in 1:length(pairs)){ 
    fs<-gsub(".vs","",pairs[i])
    pops <- unlist(strsplit(fs, "\\."))
    # Fst with actual line to highlight the differences
    df<-fst17[fst17$pop==pairs[i],]
    plots[[i]] <- ggplot(df, aes(x =midPos, y = Fst)) + 
        geom_point(size = 1, color = "gray",alpha = 0.4, shape = 1)+
        theme_minimal()+
        theme(axis.text.x=element_blank())+
        ylab("Fst\n")+ xlab("")+ 
        ggtitle(paste0(pops[1]," vs.", pops[2]))+
        geom_line(color="steelblue", size=0.2)+
        facet_wrap(~chr, ncol = 9)
}

#Same y-axis
plots2<-list()
#TB ylim=0.8
#nonTB 0.6
for (i in 1:length(pairs)){ 
    fs<-gsub(".vs","",pairs[i])
    pops <- unlist(strsplit(fs, "\\."))
    # Fst with actual line to highlight the differences
    df<-fst17[fst17$pop==pairs[i],]
    if (i %in% c(4,8,11,13,15)) ymax=0.8
    else ymax=0.6
    plots2[[i]] <- ggplot(df, aes(x =midPos, y = Fst)) + 
        geom_point(size = 1, color = "gray",alpha = 0.4, shape = 1)+
        theme_minimal()+ylim(0,ymax)+
        theme(axis.text.x=element_blank())+
        ylab("Fst\n")+ xlab("")+ 
        ggtitle(paste0(pops[1]," vs.", pops[2]))+
        geom_line(color="steelblue", size=0.2)+
        facet_wrap(~chr, ncol = 9)
}

{png(paste0("../Output/SFS/Year2017_Fst1.png"), height = 8, width = 20, res=150, units = "in")
do.call(grid.arrange, c(plots[1:6], ncol=3))
dev.off()}

{png(paste0("../Output/SFS/Year2017_Fst2.png"), height = 12, width = 20, res=150, units = "in")
do.call(grid.arrange, c(plots[7:15], ncol=3))
dev.off()}

#plot non-TB
{png(paste0("../Output/SFS/Year2017_Fst_sameYaxis.png"), height = 16, width = 20, res=150, units = "in")
do.call(grid.arrange, c(plots2[c(1,2,3,5,6,7,9,10,12,14)], ncol=3))
dev.off()}

{png(paste0("../Output/SFS/Year2017_Fst_sameYaxis2_TB.png"), height = 12, width = 20, res=150, units = "in")
do.call(grid.arrange, c(plots2[c(4,8,11,13,15)], ncol=3))
dev.off()}

contrast without TB

Contrast against TB pop

3.2 Paiwise Fst matrix

#Plot pairwise Fst values
bcxca <- round(mean(fst17$Fst[fst17$pop=="BC17.vs.CA17"]),4)
bcxpw <- round(mean(fst17$Fst[fst17$pop=="BC17.vs.PWS17"]),4)
caxpw <- round(mean(fst17$Fst[fst17$pop=="CA17.vs.PWS17"]),4)
bcxwa <- round(mean(fst17$Fst[fst17$pop=="BC17.vs.WA17"]),4)
caxwa <- round(mean(fst17$Fst[fst17$pop=="CA17.vs.WA17"]),4)
bcxss <- round(mean(fst17$Fst[fst17$pop=="BC17.vs.SS17"]),4)
bcxtb <- round(mean(fst17$Fst[fst17$pop=="BC17.vs.TB17"]),4)
ssxtb <- round(mean(fst17$Fst[fst17$pop=="SS17.vs.TB17"]),4)
caxss <- round(mean(fst17$Fst[fst17$pop=="CA17.vs.SS17"]),4)
pwxss <- round(mean(fst17$Fst[fst17$pop=="PWS17.vs.SS17"]),4)
caxtb <- round(mean(fst17$Fst[fst17$pop=="CA17.vs.TB17"]),4)
pwxtb <- round(mean(fst17$Fst[fst17$pop=="PWS17.vs.TB17"]),4)
pwxwa <- round(mean(fst17$Fst[fst17$pop=="PWS17.vs.WA17"]),4)
ssxwa <- round(mean(fst17$Fst[fst17$pop=="SS17.vs.WA17"]),4)
tbxwa <- round(mean(fst17$Fst[fst17$pop=="TB17.vs.WA17"]),4)

fst_vec <- c(0,pwxtb,ssxtb,bcxtb,tbxwa,caxtb,
             pwxtb,0,pwxss,bcxpw,pwxwa,caxpw,
             ssxtb,pwxss,0,bcxss,ssxwa,caxss,
             bcxtb,bcxpw,bcxss,0,bcxwa,bcxca,
             tbxwa,pwxwa,ssxwa,bcxwa,0,caxwa,
             caxtb,caxpw,caxss,bcxca,caxwa,0)

fst_mat = matrix(fst_vec, nrow = 6, ncol = 6)
colnames(fst_mat) <- c("TB17","PWS17","SS17","BC17","WA17","CA17")
rownames(fst_mat) <- c("TB17","PWS17","SS17","BC17","WA17","CA17")

fst_mat[lower.tri(fst_mat, diag = F)]<-NA
write.csv(fst_mat, "../Output/SFS/Fst_matrix_2017_all.csv")

# Melt the correlation matrix
melted_cormat <- melt(fst_mat, na.rm = TRUE)
melted_cormat[melted_cormat==0]<-NA
# Heatmap
melted_cormat$color<-"a"
melted_cormat$color[melted_cormat$value>=0.1]<-"b"

ggplot(data = melted_cormat, aes(Var2, Var1, fill = value))+
    geom_tile(color = "white")+
    scale_fill_gradientn(colors=c("white", "blue"), limits=c(0, (max(melted_cormat$value, na.rm=T)+0.005)),na.value="gray80", 
                         name="Fst")+
    theme_minimal()+ xlab("")+ylab("")+
    theme(axis.text.x = element_text(angle = 0, vjust = 0, 
                                     size = 12, hjust = 0.5))+
    theme(axis.text.y = element_text(size = 12))+
    coord_fixed()+
    geom_text(aes(Var2, Var1, label = value, color=color),  size = 5)+
    scale_color_manual(values=c("black", "white"), guide='none')
ggsave("../Output/SFS/Fst_matrix_2017_all.png", height = 6, width = 6, dpi=150)

3.3 Fst change over time between PWS/SS/TB

comb<-data.frame(a=c("PWS91","PWS96","PWS07","PWS17","PWS96","PWS07","PWS17","SS96","SS06","SS17"),b=c("TB91","TB96","TB06","TB17","SS96","SS06","SS17","TB96","TB06","TB17")) 

fsts<-data.frame()
for (i in 1: nrow(comb)){
    pop1<-comb[i,1]
    pop2<-comb[i,2]
    df<-read.delim(paste0("../Data/new_vcf/angsd/fromVCF/2D/fst_folded_",pop1,"_",pop2,"_50kWindow_maf00"))
    conames<-colnames(df)[2:4]
    colnames(df)[4]<-"Fst"
    colnames(df)[1:3]<-conames
    df$pop<-paste0(pop1,".vs.",pop2)
    df$ch=as.integer(gsub("chr","", df$chr))
    df<-df[order(df$ch),]
    df$loc<-1:nrow(df)
    fsts<-rbind(fsts, df)
}
write.csv(fsts,"../Output/SFS/Fst_betweenPop.csv")

re<-aggregate(fsts$Fst, by=list(fsts$pop), mean, na.rm=T)
compa<-strsplit(re$Group.1, split=".vs.")
re$pop1<-lapply(compa, "[[", 1)
re$pop2<-lapply(compa, "[[", 2)
re$year <- as.numeric(str_extract(re$pop1, "[0-9]+"))
re$year[re$year==7|re$year==6]<-2006
re$year[re$year==91]<-1991
re$year[re$year==96]<-1996
re$year[re$year==17]<-2017
re$pop1<-str_extract(re$pop1, "[aA-zZ]+")
re$pop2<-str_extract(re$pop2, "[aA-zZ]+")
re$pops<-paste0(re$pop1,"-",re$pop2)


colors2<-brewer.pal(n=8, "Set3")
"#8DD3C7" "#FFFFB3" "#BEBADA" "#FB8072" "#80B1D3" "#FDB462" "#B3DE69" "#FCCDE5"


ggplot(re, aes(x=year, y=x, color=pops, group=pops))+
    geom_point(position=position_dodge(width = 1), size=3)+
    geom_line(position=position_dodge(width = 1))+
    ylab("Fst")+xlab("Year")+
    theme_classic()+theme(legend.title = element_blank())+
    scale_color_manual(values=colors2[c(4,1,3)])
ggsave("../Output/SFS/Fst_shift_overYears_3pops.png", width = 5, height = 3, dpi=300)

4 Use Pixy to calculate pi

4.1 Steps

1. Create all sites (invariant) vcf files
   (see invariantVCF_PWS91.sh)

#Using bcftools mpileup (-r is to specify chromosome)
bcftools mpileup -f <reference.fa> -b <bamlist.txt> -r <X> | bcftools call -m -Oz -f GQ -o <output>

#Create a sample id file (required)
# Add population info to the sample sheet
sed 's/$/\tPWS91/' PWS91.txt >pws91pop.txt

#check the sample names in the vcf file
bcftools query -l PWS91_ch1.vcf.gz
# somehow, _ was replaced by .

2. Reformat the sample file (’_’ was replaced by ‘.’ in the process of making vcf files)

pops<-c("PWS91","PWS96","PWS07","PWS17")
for (i in 1: length(pops)){
    df<-read.table(paste0("../Data/pixy/",pops[i],".txt"))
    df$V1<-gsub("_",".", df$V1)
    df$V2<-pops[i]
    write.table(df, paste0("../Data/pixy/",pops[i],"pop.txt"), quote = F, col.names=F, row.names = F, sep="\t")
}

pops<-c("TB91","TB96","TB06","TB17")
for (i in 1: length(pops)){
    df<-read.table(paste0("../Data/pixy/",pops[i],".txt"))
    df$V1<-gsub("_",".", df$V1)
    df$V2<-pops[i]
    write.table(df, paste0("../Data/pixy/",pops[i],"/" ,pops[i],"pop.txt"), quote = F, col.names=F, row.names = F, sep="\t")
}

pops<-c("SS96","SS06","SS17")
for (i in 1: length(pops)){
    df<-read.table(paste0("../Data/popinfo/",pops[i],".txt"))
    df$V1<-gsub("_",".", df$V1)
    df$V2<-pops[i]
    write.table(df, paste0("../Data/pixy/",pops[i],"/" ,pops[i],"pop.txt"), quote = F, col.names=F, row.names = F, sep="\t")
}
pops<-c("CA17","BC17","WA17")
for (i in 1: length(pops)){
    df<-read.table(paste0("../Data/popinfo/",pops[i],".txt"))
    df$V1<-gsub("_",".", df$V1)
    df$V2<-pops[i]
    write.table(df, paste0("../Data/pixy/",pops[i],"/" ,pops[i],"pop.txt"), quote = F, col.names=F, row.names = F, sep="\t")
}

3. Run Pixy (example: PWS91 Chr1)

#index the vcf.gz file
tabix PWS91_ch1.vcf.gz

# Run Pixy 
#first activate the conda env (py38) -runs on older Python (<=3.8)
conda activate py38
cd ~/Projects/PacHerring/Data/pixy
pixy --stats pi --vcf PWS91_ch1.vcf.gz --populations pws91pop.txt --window_size 10000 --n_core 8 --output_prefix PWS91_ch1

conda deactivate

4. Summarize the output from Pixy for PWS91 Chr1

pipi<-read.table("../Data/pixy/PWS91/PWS91_ch1_pi.txt", header=T)
ggplot(pipi, aes(x=window_pos_1, y=avg_pi))+
    geom_line(color=blu)+xlab('')+ylab(expression("mean "*pi))
ggsave("../Output/SFS/pixy_pi_pws91_ch1_line.png", width = 6, height = 3.5, dpi=300)

ggplot(pipi, aes(x=window_pos_1, y=avg_pi))+
    geom_point(size=0.3, color="gray20")

mean(pipi$avg_pi)
# 0.00278471

#weighted mean
pipi$pi_sum<-pipi$avg_pi*pipi$no_sites
sum(pipi$pi_sum)/sum(pipi$no_sites)
# 0.002775464

4.2 Compare π estimated from ANGSD vs. Pixy

#Compare with the pi estimated from ANGSD SFS 
theta<-read.delim(paste0('../Data/new_vcf/angsd/fromBam/unfolded/PWS91_50kwin_10kstep.pestPG'))
theta$pi<-theta$tP/theta$nSites
mean(theta$pi[theta$Chr=="chr1"])
#0.003826297

#chr1 comparison
ch1<-pipi[,c("chromosome", "avg_pi")]
ch1$method<-"Pixy"
theta2<-theta[,c("Chr","pi")]
theta2$method<-"ANGSD"
colnames(ch1)[1:2]<-c("Chr","pi")
ch1<-rbind(ch1,theta2)


ggplot(ch1, aes(x=method, y=pi))+
    geom_boxplot(aes(middle=mean(pi), color=method),outlier.alpha = 0.2)+
    theme_classic()+xlab('')+ylab(expression(pi))+
    scale_color_manual(values=c("#1f78b4","#fb9a99"), guide='none')
ggsave("../Output/SFS/Pi_comparison_pixy.vs.angsd.pws91.ch1.png", width=4, height=4,dpi=200 )

4.2.1 Run Pixy for all chromosomes for PWS

#index vcf files for all chromosomes
for f in *.vcf.gz; do
filename=$(basename $f)
tabix $f 
done

#create a script to run pixy
for (j in 1: length(pops)){
    sink(paste0("../Data/pixy/runpixy_", pops[j],".sh"))
    cat("#!/bin/bash\n\n")
    for (i in 1:26){
        cat(paste0("pixy --stats pi --vcf ",pops[j],"_ch",i,".vcf.gz --populations ",pops[j],"pop.txt --window_size 10000 --n_core 8 --output_prefix ",pops[j], "_ch",i, "\n"))
    }
    sink(NULL)
}

4.2.2 Plot the outputs from Pixy for all PWS groups

#read the output file for PWS:
pops<-c("PWS91","PWS96","PWS07","PWS17")

for (p in 1 : length(pops)){
    pixy<-data.frame()
    for (i in 1:26){
        df<-read.table(paste0("../Data/pixy/",pops[p],"/",pops[p],"_ch",i,"_pi.txt"), header=T)
        df$method<-"Pixy"
        df$WinCenter<-df$window_pos_2-5000
        df<-df[,c("chromosome","WinCenter","avg_pi","method")]
        colnames(df)[c(1,3)]<-c("Chr","pi")
        pixy<-rbind(pixy, df)
    }
    write.csv(pixy, paste0("../Output/Pi/",pops[p], "_Pi_pixy_per50kWindow.csv"))
    
    #Compare with the pi estimated from ANGSD SFS 
    theta<-read.delim(paste0('../Data/new_vcf/angsd/fromBam/unfolded/',pops[p],'_50kwin_10kstep.pestPG'))
    theta$pi<-theta$tP/theta$nSites
    theta$method<-"ANGSD"
    pi<-rbind(pixy, theta[,c("Chr","WinCenter","pi","method")])

    pi$Chr<-factor(pi$Chr, levels=paste0("chr",1:26))
    ggplot(pi, aes(x=Chr, y=pi, color=method))+
        geom_boxplot(aes(middle=mean(pi)),outlier.alpha = 0.2, outlier.size = 0.6)+
        theme_classic()+theme(axis.text.x = element_text(angle=45, hjust =1))+
        xlab("")+ylab(expression(pi))+ggtitle(pops[p])+
        scale_color_manual(values=c("#1f78b4","#fb9a99"))
    ggsave(paste0("../Output/Pi/Pi_comparison_pixy.vs.angsd.",pops[p], ".png"), width = 10, height = 5, dpi=100)
    
    # genome-wide mean pi comparison
    means <- aggregate(pi ~  method, pi, mean)
    
    ggplot(pi, aes(x=method, y=pi))+
        geom_boxplot(aes(color=method), outlier.alpha = 0.2, outlier.size = 0.6)+
        theme_classic()+ ggtitle(pops[p])+
        geom_point(stat = "summary", fun = "mean", color="gray40")+
         xlab("")+ylab(expression(pi))+
        scale_color_manual(values=c("#1f78b4","#fb9a99"), guide='none')+
        geom_text(data = means, aes(label = round(pi, digits=5), y = pi + 0.02), color="gray40")
    ggsave(paste0("../Output/Pi/Pi_comparison_pixy.vs.angsd_mean_",pops[p],".png"), width = 3, height = 3, dpi=200)
}

• Pixy estimates have more variability
• Pixy esitmates are slightly lower than ANSGD estiamtes

# Assess the differences between ANGSD and Pixy
pops<-c("PWS91","PWS96","PWS07","PWS17")
diff<-data.frame(pop=pops)
for (p in 1 : length(pops)){
    pixy<-data.frame()
    for (i in 1:26){
        df<-read.table(paste0("../Data/pixy/",pops[p],"/",pops[p],"_ch",i,"_pi.txt"), header=T)
        df$method<-"Pixy"
        df$WinCenter<-df$window_pos_2-5000
        df<-df[,c("chromosome","WinCenter","avg_pi","method")]
        colnames(df)[c(1,3)]<-c("Chr","pi")
        pixy<-rbind(pixy, df)
    }
    #mean.pixy<-aggregate(pixy$pi, by=list(pixy$Chr), mean, na.rm=T)
    
    #Pi estimated from ANGSD SFS 
    theta<-read.delim(paste0('../Data/new_vcf/angsd/fromBam/unfolded/',pops[p],'_50kwin_10kstep.pestPG'))
    theta$pi<-theta$tP/theta$nSites
    theta$method<-"ANGSD"
    pi<-rbind(pixy, theta[,c("Chr","WinCenter","pi","method")])

    means<-aggregate(pi$pi, by=list(pi$method), mean, na.rm=T)
    
    #differences in pi estimates
    
    diff$prop.difference[p]<-means$x[means$Group.1=="ANGSD"]/means$x[means$Group.1=="Pixy"]
}


knitr::kable(diff)

pops<-c("PWS91","PWS96","PWS07","PWS17")
Pi<-data.frame()
for (i in 1:length(pops)){
    #pixy
    pi<-read.csv(paste0("../Output/Pi/",pops[i], "_Pi_pixy_per50kWindow.csv"), row.names =1 )
    pi$Chr<-factor(pi$Chr, levels=paste0("chr",1:26))
    pi$pop<-pops[i]
    Pi<-rbind(Pi,pi)
    
    theta<-read.delim(paste0('../Data/new_vcf/angsd/fromBam/unfolded/',pops[i],'_50kwin_10kstep.pestPG'))
    theta$pi<-theta$tP/theta$nSites
    theta$pop<-pops[i]
    theta$method<-"ANGSD"
    theta<-theta[,c("Chr", "WinCenter","pi","method","pop")]    
    Pi<-rbind(Pi, theta)
}

Pi$pop<-factor(Pi$pop, levels=pops)

# genome-wide mean pi comparison
means <- aggregate(Pi$pi, by=list(Pi$method, Pi$pop), mean, na.rm=T)
colnames(means)<-c("method","pop", "pi")
ggplot(Pi, aes(x=pop, y=pi, color=method))+
    geom_boxplot(position=position_dodge(width=0.8),  outlier.alpha = 0.2, outlier.size = 0.6)+
    theme_classic() + ylim(0,0.045)+
    stat_summary(fun = "mean", geom = "point", position=position_dodge(width=0.8), shape=16)+
    xlab("")+ylab(expression(pi))+
    scale_color_manual(values=c("#1f78b4","#fb9a99"))+
    annotate(geom="text", x=as.vector(sapply(1:4, function(x) c(x-.2, x+.2))), y=rep(c(0.018, 0.02),times=4), label=round(means$pi, digits=5), size=2.5)

ggsave(paste0("../Output/Pi/Pi_comparison_pixy.vs.angsd_mean_PWS.png"), width =6, height = 3, dpi=300)

ggplot(Pi, aes(x=pop, y=pi, color=method))+
    geom_boxplot(position=position_dodge(width=0.8),  outlier.alpha = 0.2, outlier.size = 0.6)+
    theme_classic() +ylim(0,0.015)+
    stat_summary(fun = "mean", geom = "point", position=position_dodge(width=0.8), shape=16)+
    xlab("")+ylab(expression(pi))+
    scale_color_manual(values=c("#1f78b4","#fb9a99"))
    annotate(geom="text", x=as.vector(sapply(1:4, function(x) c(x-.2, x+.2))), y=rep(c(0.018, 0.02),times=4), label=round(means$pi, digits=5), size=2)

ggsave(paste0("../Output/Pi/Pi_comparison_pixy.vs.angsd_mean_PWS_zoomed.png"), width =6.7, height = 3, dpi=300)

4.2.3 Compare across years -PWS

pops<-c("PWS91","PWS96","PWS07","PWS17")
Pi<-data.frame()
for (i in 1:length(pops)){
    pi<-read.csv(paste0("../Output/Pi/",pops[i], "_Pi_pixy_per50kWindow.csv"), row.names =1 )
    pi$Chr<-factor(pi$Chr, levels=paste0("chr",1:26))
    pi$pop<-pops[i]
    Pi<-rbind(Pi,pi)
}

Pi$pop<-factor(Pi$pop, levels=pops)

means <- aggregate(pi ~ pop,Pi, mean)
colnames(means)<-c("method","pop", "pi")
ggplot(Pi, aes(x=pop, y=pi, color=pop))+
        geom_boxplot(aes(color=pop), outlier.alpha = 0.2, outlier.size = 0.6)+
        theme_classic()+ ggtitle("PWS")+
        geom_point(stat = "summary", fun = "mean", color="gray40")+
         xlab("")+ylab(expression(pi))+
        geom_text(data = means, aes(label = round(pi, digits=5), y = pi + 0.02), color="gray40")
ggsave(paste0("../Output/Pi/Pi_pixy_mean.",pops[i],".png"), width = 4.5, height = 3, dpi=200)

#read the output file for each pops:
pops<-c("TB91","TB96","TB06","TB17")

for (p in 1 : length(pops)){
    pixy<-data.frame()
    for (i in 1:26){
        df<-read.table(paste0("../Data/pixy/",pops[p],"/",pops[p],"_ch",i,"_pi.txt"), header=T)
        df$method<-"Pixy"
        df$WinCenter<-df$window_pos_2-5000
        df<-df[,c("chromosome","WinCenter","avg_pi","method")]
        colnames(df)[c(1,3)]<-c("Chr","pi")
        pixy<-rbind(pixy, df)
    }
    write.csv(pixy, paste0("../Output/Pi/",pops[p], "_Pi_pixy_per50kWindow.csv"))
}

Pi<-data.frame()
for (i in 1:length(pops)){
    pi<-read.csv(paste0("../Output/Pi/",pops[i], "_Pi_pixy_per50kWindow.csv"), row.names =1 )

    pi$Chr<-factor(pi$Chr, levels=paste0("chr",1:26))
    ggplot(pi, aes(x=Chr, y=pi))+
        geom_boxplot(aes(middle=mean(pi)),outlier.alpha = 0.2, outlier.size = 0.6,color="#1f78b4")+
        theme_classic()+theme(axis.text.x = element_text(angle=45, hjust =1))+
        xlab("")+ylab(expression(pi))+ggtitle(pops[i])
    ggsave(paste0("../Output/Pi/Pi_pixy.perChrom.",pops[i], ".png"), width = 8.5, height = 5, dpi=300)
    
    pi$pop<-pops[i]
    Pi<-rbind(Pi,pi)
}

Pi$pop<-factor(Pi$pop, levels=pops)
means <- aggregate(pi ~ pop,Pi, mean)
ggplot(Pi, aes(x=pop, y=pi, color=pop))+
        geom_boxplot(aes(color=pop), outlier.alpha = 0.2, outlier.size = 0.6)+
        theme_classic()+ ggtitle("TB")+
        geom_point(stat = "summary", fun = "mean", color="gray40")+
         xlab("")+ylab(expression(pi))+
        geom_text(data = means, aes(label = round(pi, digits=5), y = pi + 0.02), color="gray40")
ggsave(paste0("../Output/Pi/Pi_pixy_mean.TB.png"), width = 4.5, height = 3, dpi=200)

pops<-c("PWS91","PWS96","PWS07","PWS17","TB91","TB96","TB06","TB17","SS96","SS06","SS17")
year<-c(1991,1996,2007,2017,1991,1996,2006,2017,1996,2006,2017)
meanPi<-data.frame(pop.yr=pops, year=year)
for (i in 1:length(pops)){
    df<-read.csv(paste0("../Output/Pi/",pops[i], "_Pi_pixy_per50kWindow.csv"), row.names =1 )
    meanPi$mean[i]<-mean(df$pi, na.rm=T)
    meanPi$pop[i]<-gsub("\\d.+","",pops[i])
}


ggplot(meanPi, aes(x=year, y=mean, color=pop))+
    geom_point(size=3)+
    geom_line()+
    xlab("")+ylab(paste0("Mean ",expression(pi)))+
    theme_linedraw()+
    scale_color_manual(values=cols[c(2,1,5)])
ggsave("../Output/Pi/Pi_overYears.PWS.TB.SS.png", width = 6, height = 4, dpi=300)

4.2.4 Bootstrap pi values to get 95% CI

# average theta values across sites

#############
# Parameters
#############
# number of chromosomes in each sample
nchr=26
nboot <- 1000

####################
# load functions
require(data.table)
require(boot) # for bootstrap CIs


# For Tajima's D calcs. After https://github.com/ANGSD/angsd/blob/master/misc/stats.cpp
a1f <- function(nsam) return(sum(1/seq(1, nsam-1)))
a2f <- function(nsam) return(sum(1/(seq(1, nsam-1)*seq(1, nsam-1))))
b1f <- function(nsam) return((nsam + 1)/(3*(nsam-1)))
b2f <- function(nsam) return((2*(nsam*nsam + nsam + 3))/(9*nsam*(nsam - 1)))
c1f <- function(a1, b1) return(b1 - (1/a1))
c2f <- function(nsam, a1, a2, b2) return(b2 - ((nsam + 2)/(a1*nsam)) + (a2/(a1 * a1)))
e1f <- function(a1, c1) return(c1/a1)
e2f <- function(a1, a2, c2) return(c2/((a1*a1) + a2))

# Tajima's D calculation
# nsam: sample size
# thetaW: Watterson's theta (# segregating sites / a1)
# sumk: theta pi (average number of SNPs in pairwise comparisons)
# after https://github.com/ANGSD/angsd/blob/master/misc/stats.cpp
tajd <- function(nsam, thetaW, sumk){
    a1 <- a1f(nsam)
    segsites <- thetaW * a1
    if(segsites == 0) return(0)
    a2 <- a2f(nsam)
    b1 <- b1f(nsam)
    b2 <- b2f(nsam)
    c1 <- c1f(a1, b1)
    c2 <- c2f(nsam, a1, a2, b2)
    e1 <- e1f(a1, c1)
    e2 <- e2f(a1, a2, c2)
    res <- (sumk - (thetaW))/sqrt((e1*segsites) + ((e2*segsites)*(segsites-1)))
    return(res)
}

# calc thetas
calcthetas <- function(dat, nchr, nloci){
    # calc ave theta
    thetas <- as.numeric(dat[, .(tW = sum(exp(Watterson)/nloci, na.rm = TRUE), tP = sum(exp(Pairwise)/nloci, na.rm = TRUE))])

    # calculate Tajima's D
    thetas[3] <- tajd(nchr, thetas[1], thetas[2])
    
    #return
    names(thetas) <- c('tW', 'tP', 'tD')
    return(thetas)
}

# calculate stats from specified LGs for block bootstrapping across LGs
thetablock <- function(lgs, indices, alldata, nchr, regs){
    # make bootstrapped dataset
    mydata <- do.call("rbind", lapply(indices, function(n) subset(alldata, Chromo==lgs[n])))
    
    # calculate number of callable loci, given the LGs in this bootstrapped sample
    nloci <- regs[Chromo %in% lgs, .(len = Pos2 - Pos1 + 1), by = .(Chromo, Pos1)][, sum(len)] # sum of bp in the callable region
    
    # calc thetas
    thetas <- calcthetas(mydata, nchr, nloci)
    
    # return
    return(thetas)
}

#using window based angsd file (use preknown loci in 50000 windows)
thetablock2 <- function(lgs, indices, alldata, nchr, regs){
    # make bootstrapped dataset
    mydata <- do.call("rbind", lapply(indices, function(n) subset(alldata, Chromo==lgs[n])))
    
    # calculate number of callable loci, given the LGs in this bootstrapped sample
    #nloci <- regs[Chromo %in% lgs, .(len = Pos2 - Pos1 + 1), by = .(Chromo, Pos1)][, sum(len)] # sum of bp in the callable region

    nloci <- alldata[Chromo %in% lgs,sum(nSites)] # sum of bp in the callable region
    
    # calc thetas
    thetas <- calcthetas(mydata, nchr, nloci)
    
    # return
    return(thetas)
}


######################
# Load data
######################
alldata<-dat
# load all loci theta calcs
dat<-fread('../Data/new_vcf/angsd/fromVCF/BC17_maf00.thetas50kWindow.gz.pestPG')
setnames(dat, 'Chr', 'Chromo')

datCan40 <- fread('analysis/thetas.Can_40.pestPG.gz')
datCan14 <- fread('analysis/thetas.Can_14.pestPG.gz')
datLof07 <- fread('analysis/thetas.Lof_07.pestPG.gz')
datLof11 <- fread('analysis/thetas.Lof_11.pestPG.gz')
datLof14 <- fread('analysis/thetas.Lof_14.pestPG.gz')

# gatk loci
datCan40gatk <- fread('analysis/thetas.Can_40.gatk.pestPG.gz')
datCan14gatk <- fread('analysis/thetas.Can_14.gatk.pestPG.gz')
datLof07gatk <- fread('analysis/thetas.Lof_07.gatk.pestPG.gz')
datLof11gatk <- fread('analysis/thetas.Lof_11.gatk.pestPG.gz')
datLof14gatk <- fread('analysis/thetas.Lof_14.gatk.pestPG.gz')

# fix name
setnames(datCan40, '#Chromo', 'Chromo')
setnames(datCan14, '#Chromo', 'Chromo')
setnames(datLof07, '#Chromo', 'Chromo')
setnames(datLof11, '#Chromo', 'Chromo')
setnames(datLof14, '#Chromo', 'Chromo')

setnames(datCan40gatk, '#Chromo', 'Chromo')
setnames(datCan14gatk, '#Chromo', 'Chromo')
setnames(datLof07gatk, '#Chromo', 'Chromo')
setnames(datLof11gatk, '#Chromo', 'Chromo')
setnames(datLof14gatk, '#Chromo', 'Chromo')

windownames<-colnames(dat)[1]
colnames(dat)[1]<-"window"
## list of callable regions

dat$window<-gsub("\\(",'', dat$window)
dat$window<-gsub("\\)$",'', dat$window)
dat$window<-gsub("\\)",',', dat$window)
windows1<-str_split_fixed(dat$window,",",6) 
colnames(windows1)<-c('IndexStart', 'IndexStop','firstPos_withData','lastPos_withData','WinStart','WinStop')

dat<-cbind(windows1, dat)

regs<-dat[,c("Chromo","WinStart","WinStop")]
names(regs)<-c('Chromo', 'Pos1', 'Pos2')
#regs <- fread('data_2020.05.07/Callable_bases_gadmor2.bed')
#setnames(regs, c('Chromo', 'Pos1', 'Pos2'))
#
## list of no damage sites
#nodam <- fread('data_2020.05.07/GATK_filtered_SNP_no_dam2.tab')
#setnames(nodam, c('Chromo', 'Pos', 'REF', 'ALT'))
#
#
## remove unplaced
#datCan40 <- datCan40[grep('Unplaced', Chromo, invert = TRUE), ]
#datCan14 <- datCan14[grep('Unplaced', Chromo, invert = TRUE), ]
#datLof07 <- datLof07[grep('Unplaced', Chromo, invert = TRUE), ]
#datLof11 <- datLof11[grep('Unplaced', Chromo, invert = TRUE), ]
#datLof14 <- datLof14[grep('Unplaced', Chromo, invert = TRUE), ]
#
#datCan40gatk <- datCan40gatk[grep('Unplaced', Chromo, invert = TRUE), ]
#datCan14gatk <- datCan14gatk[grep('Unplaced', Chromo, invert = TRUE), ]
#datLof07gatk <- datLof07gatk[grep('Unplaced', Chromo, invert = TRUE), ]
#datLof11gatk <- datLof11gatk[grep('Unplaced', Chromo, invert = TRUE), ]
#datLof14gatk <- datLof14gatk[grep('Unplaced', Chromo, invert = TRUE), ]

regs <- regs[Chromo != 'Unplaced', ]
nodam <- nodam[Chromo != 'Unplaced', ]

###################################################
# create table of loci trimmed to no damage sites
###################################################

datCan40gatknd <- merge(datCan40, nodam[, .(Chromo, Pos)], by = c('Chromo', 'Pos')) # trim
datCan14gatknd <- merge(datCan14, nodam[, .(Chromo, Pos)], by = c('Chromo', 'Pos')) # trim
datLof07gatknd <- merge(datLof07, nodam[, .(Chromo, Pos)], by = c('Chromo', 'Pos')) # trim
datLof11gatknd <- merge(datLof11, nodam[, .(Chromo, Pos)], by = c('Chromo', 'Pos')) # trim
datLof14gatknd <- merge(datLof14, nodam[, .(Chromo, Pos)], by = c('Chromo', 'Pos')) # trim


################################
# Run theta calculations
################################
#nloci <- regs[, .(len = Pos2 - Pos1 + 1), by = .(Chromo, Pos1)][, sum(len)] # sum of bp in the callable region

nloci<-dat$nSites

# all loci
calcthetas(dat, nchr, nloci)
calcthetas(datCan14, nchrCan14, nloci)
calcthetas(datLof07, nchrLof07, nloci)
calcthetas(datLof11, nchrLof11, nloci)
calcthetas(datLof14, nchrLof14, nloci)

# gatk loci
calcthetas(datCan40gatk, nchrCan40, nloci)
calcthetas(datCan14gatk, nchrCan14, nloci)
calcthetas(datLof07gatk, nchrLof07, nloci)
calcthetas(datLof11gatk, nchrLof11, nloci)
calcthetas(datLof14gatk, nchrLof14, nloci)

# gatk no damage loci
calcthetas(datCan40gatknd, nchrCan40, nloci)
calcthetas(datCan14gatknd, nchrCan14, nloci)
calcthetas(datLof07gatknd, nchrLof07, nloci)
calcthetas(datLof11gatknd, nchrLof11, nloci)
calcthetas(datLof14gatknd, nchrLof14, nloci)




# block bootstrapping across LGs

#lgs <- datCan40[, sort(unique(Chromo))]
datlist <- list(datCan40, datCan14, datLof07, datLof11, datLof14, 
                datCan40gatk, datCan14gatk, datLof07gatk, datLof11gatk, datLof14gatk,
                datCan40gatknd, datCan14gatknd, datLof07gatknd, datLof11gatknd, datLof14gatknd)
names(datlist) <- c('Can40 all loci', 'Can14 all loci', 'Lof07 all loci', 'Lof11 all loci', 'Lof14 all loci', 
                    'Can40 gatk loci', 'Can14 gatk loci', 'Lof07 gatk loci', 'Lof11 gatk loci', 'Lof14 gatk loci',
                    'Can40 gatk no dam loci', 'Can14 gatk no dam loci', 'Lof07 gatk no dam loci', 'Lof11 gatk no dam loci', 
                    'Lof14 gatk no dam loci')
nchrlist <- list(nchrCan40, nchrCan14, nchrLof07, nchrLof11, nchrLof14, nchrCan40, nchrCan14, nchrLof07, nchrLof11, nchrLof14, nchrCan40, nchrCan14, nchrLof07, nchrLof11, nchrLof14)

thetabootout <- data.frame(type = names(datlist), tW = NA, tWl95 = NA, tWu95 = NA, tP = NA, tPl95 = NA, tPu95 = NA, tD = NA, tDl95 = NA, tDu95 = NA)


#####

lgs <- dat[, sort(unique(Chromo))]

bootlg <- boot(lgs, thetablock2, nboot,  alldata = dat, nchr = nchr, regs = regs)


for(i in 1:length(datlist)){
    print(names(datlist)[i])
    
    
    bootlg <- boot(lgs, thetablock, nboot,  alldata = datlist[[i]], nchr = nchrlist[[i]], regs = regs)
    
    print(bootlg)
    ciW <- boot.ci(bootlg, type = c('perc'), index = 1)
    ciP <- boot.ci(bootlg, type = c('perc'), index = 2)
    ciD <- boot.ci(bootlg, type = c('perc'), index = 3)
    
    thetabootout$tW[i] <- bootlg$t0[1] # the point estimates
    thetabootout$tP[i] <- bootlg$t0[2]  
    thetabootout$tD[i] <- bootlg$t0[3]

    thetabootout$tWl95[i] <- ciW$percent[4] # the confidence intervals
    thetabootout$tWu95[i] <- ciW$percent[5]

    thetabootout$tPl95[i] <- ciP$percent[4]
    thetabootout$tPu95[i] <- ciP$percent[5]

    thetabootout$tDl95[i] <- ciD$percent[4]
    thetabootout$tDu95[i] <- ciD$percent[5]
}

# save
write.csv(thetabootout, file = 'analysis/thetas.boot.cis.csv')

5 Fst between populations in 1991, 1996 and 2006/7

# 1. Y1996
pops<-c("PWS96","SS96","TB96")
comb<-t(combn(pops,2))

fst<-data.frame()
for (i in 1: nrow(comb)){
    pop1<-comb[i,1]
    pop2<-comb[i,2]
    df<-read.delim(paste0("../Data/new_vcf/angsd/fromVCF/2D/fst_folded_",pop1,"_",pop2,"_50kWindow_maf00"))
    conames<-colnames(df)[2:4]
    colnames(df)[4]<-"Fst"
    colnames(df)[1:3]<-conames
    df$pop<-paste0(pop1,".vs.",pop2)
    df$ch=as.integer(gsub("chr","", df$chr))
    df<-df[order(df$ch),]
    df$loc<-1:nrow(df)
    fst<-rbind(fst, df)
}

evens<-paste0("chr",seq(2,26, by=2))
#Plot Fst values across Genome
fst$color<-"col1"
fst$color[fst$chr %in% evens]<-"col2"
fst$pop<-factor(fst$pop, levels=unique(fst$pop))

#add chromosome number
df<-fst[fst$pop=="PWS96.vs.SS96",]
rows<-data.frame(chr=1:26)
for (i in 1:26){
    if (i ==1){
        rows$n[i]<-nrow(df[df$ch==i,])
        rows$middle[i]<-nrow(df[df$ch==i,])/2
    }
    if (i >1){
        rows$n[i]<-nrow(df[df$ch==i,])
        rows$middle[i]<-sum(rows$n[1:(i-1)])+rows$n[i]/2
    }
}

ggplot(fst, aes(x=loc, y=Fst, color=color))+
    facet_wrap(~pop, ncol = 1, strip.position="right")+
    geom_point(size=0.2, alpha=0.6)+
    scale_color_manual(values=c("gray60","steelblue"))+
    theme_bw()+ggtitle("1996")+
    ylab("Fst")+xlab('Genome position')+
    theme(legend.position = "none", panel.grid.major.x = element_blank(), panel.grid.minor.x = element_blank())+
    scale_x_continuous(breaks=rows$middle, labels=1:26)
ggsave("../Output/SFS/Y1996_Fst_pairwise_comparison.png", width = 16, height = 7, dpi=300)


compare<-unique(fst$pop)
pairfst<-data.frame(matrix(ncol=3, nrow=3), row.names=c("TB96","PWS96","SS96"))
colnames(pairfst)<-c("TB96","PWS96","SS96")
for (i in 1:3){
    pop1<-comb[i,1]
    pop2<-comb[i,2]
    df<-fst[fst$pop==compare[i],]
    pairfst[pop1,pop2]<-mean(df$Fst, na.rm=T)
    pairfst[pop2,pop1]<-mean(df$Fst, na.rm=T)
}
write.csv(pairfst,"../Output/SFS/Y1996_pairwiseFst_matrix.csv")

#df<-read.csv("../Output/SFS/Y1996_pairwiseFst_matrix.csv", row.names = 1)
df<-pairfst
diag(df)<-0
df[lower.tri(df)]<-NA
df$pop<-rownames(df)
dfm<-melt(df,na.rm=T, id.vars='pop')
#NA to diagonal
dfm$value[dfm$value==0]<-NA
dfm$pop<-factor(dfm$pop, levels=c("TB96","PWS96","SS96"))
dfm$value<-round(dfm$value, 4)
ggplot(data = dfm, aes(pop, variable, fill = value))+
    geom_tile(color = "white")+
    scale_fill_gradientn(colors=c("white", "#0C54FFCC"), limits=c(0, (max(dfm$value, na.rm=T)+0.05)),na.value="gray80", 
                         name="Fst")+
    theme_minimal()+ xlab("")+ylab("")+
    theme(axis.text.x = element_text(angle = 0, vjust = 0, 
                                     size = 12, hjust = 0.5))+
    theme(axis.text.y = element_text(size = 12))+
    coord_fixed()+
    geom_text(aes(pop, variable, label = value), color = "black", size = 5)
ggsave(paste0("../Output/SFS/Y1996_pairwiseFst.png"), width = 5, height = 5, dpi=300)

# 2. Y2006/2007
pops<-c("PWS07","SS06","TB06")
comb<-t(combn(pops,2))

fst<-data.frame()
for (i in 1: nrow(comb)){
    pop1<-comb[i,1]
    pop2<-comb[i,2]
    df<-read.delim(paste0("../Data/new_vcf/angsd/fromVCF/2D/fst_folded_",pop1,"_",pop2,"_50kWindow_maf00"))
    conames<-colnames(df)[2:4]
    colnames(df)[4]<-"Fst"
    colnames(df)[1:3]<-conames
    df$pop<-paste0(pop1,".vs.",pop2)
    df$ch=as.integer(gsub("chr","", df$chr))
    df<-df[order(df$ch),]
    df$loc<-1:nrow(df)
    fst<-rbind(fst, df)
}

#Plot Fst values across Genome
fst$color<-"col1"
fst$color[fst$chr %in% evens]<-"col2"
fst$pop<-factor(fst$pop, levels=unique(fst$pop))

ggplot(fst, aes(x=loc, y=Fst, color=color))+
    facet_wrap(~pop, ncol = 1, strip.position="right")+
    geom_point(size=0.2, alpha=0.6)+
    scale_color_manual(values=c("gray60","steelblue"))+
    theme_bw()+
    ylab("Fst")+xlab('Genome position')+
    theme(legend.position = "none", panel.grid.major.x = element_blank(), panel.grid.minor.x = element_blank())+ggtitle("2006/07")+
    scale_x_continuous(breaks=rows$middle, labels=1:26)
ggsave("../Output/SFS/Y2006_Fst_pairwise_comparison.png", width = 16, height = 7, dpi=300)


compare<-unique(fst$pop)
pairfst<-data.frame(matrix(ncol=3, nrow=3), row.names=c("TB06","PWS07","SS06"))
colnames(pairfst)<-c("TB06","PWS07","SS06")
for (i in 1:3){
    pop1<-comb[i,1]
    pop2<-comb[i,2]
    df<-fst[fst$pop==compare[i],]
    pairfst[pop1,pop2]<-mean(df$Fst, na.rm=T)
    pairfst[pop2,pop1]<-mean(df$Fst, na.rm=T)
}
write.csv(pairfst,"../Output/SFS/Y2006_pairwiseFst_matrix.csv")

df<-pairfst
diag(df)<-0
df[lower.tri(df)]<-NA
df$pop<-rownames(df)
dfm<-melt(df,na.rm=T, id.vars='pop')
#NA to diagonal
dfm$value[dfm$value==0]<-NA
dfm$pop<-factor(dfm$pop, levels=c("TB06","PWS07","SS06"))
dfm$value<-round(dfm$value, 4)
ggplot(data = dfm, aes(pop, variable, fill = value))+
    geom_tile(color = "white")+
    scale_fill_gradientn(colors=c("white", "#0C54FFCC"), limits=c(0, (max(dfm$value, na.rm=T)+0.05)),na.value="gray80", 
                         name="Fst")+
    theme_minimal()+ xlab("")+ylab("")+
    theme(axis.text.x = element_text(angle = 0, vjust = 0, 
                                     size = 12, hjust = 0.5))+
    theme(axis.text.y = element_text(size = 12))+
    coord_fixed()+ggtitle("2006/07")+
    geom_text(aes(pop, variable, label = value), color = "black", size = 5)
ggsave(paste0("../Output/SFS/Y2006_pairwiseFst.png"), width = 5, height = 5, dpi=300)


#2017




f17<-read.csv("../Output/SFS/Fst_matrix_2017_all.csv")
f17<-f17[1:3,1:4]
# Melt the correlation matrix
f17m <- melt(f17, na.rm = TRUE)
f17m[f17m==0]<-NA

colnames(f17m)[1:3]<-c("P1","P2","fst")
f17m$P1<-factor(f17m$P1, levels=c("TB17","PWS17","SS17"))

ggplot(f17m, aes(P1, P2, fill = fst))+
    geom_tile(color = "white")+
    scale_fill_gradientn(colors=c("white", "#0C54FFCC"), limits=c(0, (max(f17m$fst, na.rm=T)+0.05)),na.value="gray80",  name="Fst")+
                        
    theme_minimal()+ xlab("")+ylab("")+
    theme(axis.text.x = element_text(angle = 0, vjust = 0, 
                                     size = 12, hjust = 0.5))+
    theme(axis.text.y = element_text(size = 12))+
    coord_fixed()+
    geom_text(aes(P1, P2, label = fst),  size = 5)
ggsave("../Output/SFS/Y2017_pairwiseFst.png", height = 5, width = 5, dpi=150)

# 3. Y1991 

df<-read.delim(paste0("../Data/new_vcf/angsd/fromVCF/2D/fst_folded_PWS91_TB91_50kWindow_maf00"))
conames<-colnames(df)[2:4]
colnames(df)[4]<-"Fst"
colnames(df)[1:3]<-conames
df$pop<-paste0("PWS91.vs.TB91")
df$ch=as.integer(gsub("chr","", df$chr))
df<-df[order(df$ch),]
df$loc<-1:nrow(df)
fst<-df
#Plot Fst values across Genome
fst$color<-"col1"
fst$color[fst$chr %in% evens]<-"col2"
fst$pop<-factor(fst$pop, levels=unique(fst$pop))

ggplot(fst, aes(x=loc, y=Fst, color=color))+
    facet_wrap(~pop, ncol = 1, strip.position="right")+
    geom_point(size=0.2, alpha=0.6)+
    scale_color_manual(values=c("gray60","steelblue"))+
    theme_bw()+
    ylab("Fst")+xlab('Genome position')+
    theme(legend.position = "none", panel.grid.major.x = element_blank(), panel.grid.minor.x = element_blank())+ggtitle("1991")+
    scale_x_continuous(breaks=rows$middle, labels=1:26)+
    annotate("text", x=1, y=0.7, label="Mean Fst = 0.142", hjust=0)
    
ggsave("../Output/SFS/Y1991_Fst_pairwise_comparison.png", width = 16, height = 2.5, dpi=300)

pairfst<-mean(fst$Fst, na.rm=T)

pairfst
#0.1417637